Professional Documents
Culture Documents
2
Analysis of Amino Acids by Paper Chromatography
Esconde, Yvonne Keithlene J. BS Nursing 1A
Grp. 3 / Thurs (7:00–7:30 PM) Aug. 23, 2018
Rating: ________
I. Objectives
1. To separate amino acids using the technique of paper chromatography
2. To identify the color reactions of different amino acids with the detection reagent
II. Introduction
Chromatography is a technique for analyzing or separating mixtures of substances into
their components. There are various forms of chromatography techniques. Most of
these techniques involve two distinct phases: the stationary phase and the mobile
phase. In paper chromatography, the stationary phase is a liquid which is absorbed on
the cellulose paper, whereas the mobile phase is a mixture of solvents suitable for
separating the components in a sample. The relative affinity of a substance for each
phase depends on properties such as molecular weight, structure and shape of the
molecule, and the polarity of the molecule.
In this experiment, very small volumes of solutions containing amino acids will be
applied using capillary tubes. After the solutions have been applied, the paper will be
placed inside the chromatographic chamber for development. As the mobile phase rises
on the paper, it will eventually encounter the “spots” of amino acids. The fate of each
amino acid in the mixture now depends on the affinity of each substance for the mobile
and stationary phases. It is these differences in amino acid affinities that lead to the
separation. Identification of the amino acid is done by spraying with Ninhydrin
solution. The Ninhydrin forms a blue violet complex with an amino acid except for
proline/hydroxyl proline which gives a yellow color complex.
Amino acids play central roles both as building blocks of proteins and as intermediates
in metabolism. The 20 amino acids that are found within proteins convey a vast array
of chemical versatility.
In this experiment, students will identify amino acids by qualitative analysis and paper
chromatography.
III. Materials
A. Equipment
• (1) 250-mL • Whatman #1 Paper
Beaker
• (1) Capillary Tube • Gloves
• Aluminum Foil • (5) 10-mL Test Tubes
B. Reagents
• Solvent System [1-butanol, acetic acid, water (12:3:5)]
• (1 mg/mL) Alanine • (1 mg/mL) Tyrosine
• (1 mg/mL) Leucine • (1 mg/mL) Arginine
• (1 mg/mL) Lysine • (1 mg/mL) Glycine
• (1 mg/mL) Cystein • (4 mg/mL) Ninhydrin Solution
• (1 mg/mL) • Unknown amino acid mixture
Tryptophan
IIII. Methodology (Schematic Diagram)
A. Preparation of Detection Reagent
• Prepare 100 mL Ninhydrin solution with a concentration of 2 mg/mL.
B. Preparation of Amino Acid Solution
• Prepare 20 mL (1 mg/mL) solution for each of the amino acid.
C. Qualitative Analysis of Amino Acids
1. Test for Amines
2. Test for Amides
3. Test for substituted Benzene Rings
4. Test for Thiol groups
5. Paper Chromatography
5. Give the structure of Ninhydrin.
IX. References
Leluk, J. (n.d.). Why Cysteine is special? Retrieved
from http://www.cryst.bbk.ac.uk/pps97/assignments/projects/leluk/project.htm
Santa Cruz Biotechnology. (2018). Ninhydrin reagent, 2% solution [photograph]. Retrieved
fromhttps://www.scbt.com/scbt/product/ninhydrin-reagent-2-solution
X. Certification / Conforme